Direct immobilization of DNA oligomers onto the amine-functionalized glass surface for DNA microarray fabrication through the activation-free reaction of oxanine

Abstract

Oxanine having an O-acylisourea structure was explored to see if its reactivity with amino group is useful in DNA microarray fabrication. By the chemical synthesis, a nucleotide unit of oxanine (Oxa-N) was incorporated into the 5′-end of probe DNA with or without the -(CH2)n- spacers (n = 3 and 12) and found to immobilize the probe DNA covalently onto the NH2-functionalized glass slide by one-pot reaction, producing the high efficiency of the target hybridization. The methylene spacer, particularly the longer one, generated higher efficiency of the target recognition although there was little effect on the amount of the immobilized DNA oligomers. The post-spotting treatment was also carried out under the mild conditions (at 25 or 42°C) and the efficiencies of the immobilization and the target recognition were evaluated similarly, and analogous trends were obtained. It has also been determined under the mild conditions that the humidity and time of the post-spotting treatment, pH of the spotting solution and the synergistic effects with UV-irradiation largely contribute to the desired immobilization and resulting target recognition. Immobilization of DNA oligomer by use of Oxa-N on the NH2-functionalized surface without any activation step would be employed as one of the advanced methods for generating DNA-conjugated solid surface.

Gold nanoparticles assisted characterization of amine functionalized polystyrene multiwell plate and glass slide surfaces

“…APTES functionalized PS plates and GS are used in the immobilization of antibodies for the detection of antigens. Similarly, oligonucleotide probes/DNA were used to detect target DNA sequences and single nucleotide polymorphisms (SNP) typing and assaying in the protein kinases activity (Dixit et al 2010;Qin et al 2007;Pack et al 2007;Hirayama et al 1996;Nikiforov et al 1994;Kim et al 2008).…”

T7 RNA Polymerases Backed up by Covalently Trapped Proteins Catalyze Highly Error Prone Transcription

“…Preparation of 60-mer oligonucleotides containing oxanine and fluorescein (FLU) was reported previously (7,9,30,31). Proteins used for DPCs were platelet factor-4 (PLA), ␤-endorphin (END), midkine (MID), histone H2A (H2A), and hNEIL1 glycosylase (NEI) (supplemental Table S1).…”

Investigation and Development of Quantum Dot-Encoded Microsphere Bioconjugates for DNA Detection by Flow Cytometry

“…A maximum of 11% nonspecific binding was detected at saturation (Additional file 1). Non-specific binding with high concentrations of fluorescent probe could illustrate the natural tendency of cyanine fluorophores to react with polymer surfaces without activators [16], and/or the interaction of QDEM carboxyl groups with the amino groups carried by the oligonucleotides [24]. Non-specific binding was limited by storing the QDEMs in a buffer containing 1% bovine serum albumin (BSA).…”

“…Non-specific binding was limited by storing the QDEMs in a buffer containing 1% bovine serum albumin (BSA). Native BSA has shown useful blocking agent properties in microsphere covalent binding assays by covering hydrophobic surface and avoiding non-specific interaction [24]. The QDEM conjugation assay was highly specific and sensitive with the optimal conditions of 65 pmol of a 6 C amino-modified conjugation probe and 1 h incubation in imidazole buffer.…”